The invention relates generally to the fields of molecular biology, biochemistry, plant pathology, and agriculture. More particularly, the invention relates to proteins and polynucleotides associated with resistance to microbial plant pathogens.
Disease resistance in plants is often controlled by host recognition of specific pathogen determinants. Resistance (R) gene products in a host plant are believed to function as receptors that recognize effector proteins produced directly or indirectly by a pathogen""s avirulence genes. Over the past decade, a number of dominant R genes have been characterized from diverse plants. The proteins encoded by these genes can be grouped into several classes based on structure: serine/threonine kinases, proteins with a nucleotide binding site and leucine-rich repeats (NBS-LRR), presumed extracellular LRR-containing proteins with or without a transmembrane domain, and serine/threonine receptor-like kinases. Baker et al., Science 276, 726 (1997); Staskawicz et al., Science 268, 661 (1995); and Wang et al., Plant Cell 10, 765 (1998). Based on their structures, such proteins are thought to mediate their function by modulating intracellular signaling pathways.
The action of R genes is usually associated with a number of defense responses. One such response common in many different plant types is the hypersensitive response (HR). HR is characterized by rapid cell death at the site of the infection that can often be visualized as a dry brown lesion. While the intracellular signaling events that mediate resistance and HR are not completely characterized, it has been hypothesized that recognition of a pathogen-produced effector protein induces a negative regulatory pathway that can degrade the R protein. Such a pathway would regulate the intensity and duration of cell death and other elicited intracellular signals that contribute to the resistance response. Boyes et al., Proc. Natl. Acad. Sci. U.S.A. 95, 15849 (1998); S. G. Moller and N.-H. Chua, J. Mol. Biol. 293, 219 (1999).
One possible molecular mechanism for the negative regulatory pathway is ubiquitin-mediated protein degradation, which plays an important role in controlling the abundance of numerous short-lived proteins. A. Hershko and A. Ciechanover, Annu. Rev. Biochem. 67, 425 (1998); A. Ciechanover, EMBO J. 17, 7151 (1998). In this process, ubiquitin is activated by the ubiquitin-activating enzyme E1, transferred to the ubiquitin-conjugating enzyme E2, and finally linked to a target substrate by the ubiquitin ligase E3. Id. Upon attachment by ubiquitin, the target protein is subjected to degradation by the 26S proteasome. The specificity of the degradation pathway is determined by E3, which binds to the targeted substrate. Characterization of a number of E3s in animal systems indicates that a zinc-binding domain, RING (for Really Interesting New Gene) finger, is essential for many ubiquitin-mediated protein degradation processes. P. S. Freemont, Curr. Biol. 10, R84 (2000); R. J. Deshaies, Annu. Rev. Cell Dev. Biol. 15, 435 (1999).
A model for studying R-mediated plant pathogen resistance is that of rice (Oryza sativa) and the bacterial pathogen Xanthomonas oryzae, pv. oryzae (Xoo). In this system, the rice R gene Xa21 confers resistance to bacterial blight disease caused by Xoo. Transgenic cell lines expressing the protein encoded by Xa21, i.e. XA21, respond with cell death after inoculation with an avirulent strain of Xoo such as Philippine race 6 (strain pXO99 AZ), but not with virulent strain of Xoo such as Korean race 1 (strain DY890931). He et al., Science 288:2360 (2000). Although XA21 is known to be a receptor-like kinase with serine/threonine specificity [U.S. Pat. No. 5,952,485; and Song et al., Science 270, 661 (1995)], the mechanisms by which it mediates resistance and cell death are not completely understood. Identification of molecules that interact with and/or modify XA21 should help clarify these mechanisms, and provide new tools for engineering broad-spectrum, durable disease resistance in rice and other crop plants.
The invention relates to the discovery of Xb3, a polynucleotide encoding XB3 (for XA21 Binding Protein 3), a protein that interacts with the XA21 kinase. Xb3 was identified in a yeast two-hybrid assay in which a rice cDNA library was screened using the XA21 kinase as bait. The cloned Xb3 was subsequently sequenced. Based on the nucleotide sequence, it was determined that Xb3 encodes a 450 amino acid protein (i.e., XB3) that has a myristoylation site, 8 imperfect copies of ankyrin repeats, and a RING finger motif. Functional studies indicated that XB3 is a substrate for the XA21 serine/threonine kinase, and binds XA21 via its ankyrin repeat domain. Other studies indicated that XB3""s RING finger domain ubiquitinates itself, and is required for ubiquitination of XA21. In vivo protein assays indicated that XA21 is rapidly degraded in response to infection with an avirulent strain of Xoo, but not with a virulent strain. Taken together, these results suggest that XB3 serve as an E3 that negatively regulates pathogen resistance and cell death through ubiquitin-mediated protein degradation of XA21.
Accordingly, the invention features a purified nucleic acid that includes a nucleotide sequence which encodes a naturally occurring protein that: (a) shares at least 80% sequence identity with SEQ ID NO:2 and (b) has at least one functional activity of native XB3. For example, nucleic acids having a nucleotide sequence whose complement hybridizes under high stringency conditions to the nucleotide sequence of SEQ ID NO:1; those encoding a protein having the amino acid sequence of SEQ ID NO:2; and those encoding a protein that specifically binds to XA21 are featured in the invention.
In another aspect, the invention features a vector including a nucleic acid of the invention. The vector can have a nucleic acid operably linked to one or more expression control sequences. Also within the invention is a cell containing a nucleic acid of the invention.
Proteins that include an amino acid sequence that shares at least 80% sequence identity with SEQ ID NO:2 and have at least one functional activity of native XB3 (e.g., purified protein whose amino acid sequence is SEQ ID NO:2) are included in the invention. Also featured are purified proteins containing one or more of amino acid residues 1-10, 11-305, and/or 319-385 of SEQ ID NO:2; as well as fusion proteins containing one of the foregoing proteins fused to a heterologous polypeptide.
Within the invention is a purified antibody that specifically binds to a protein of the invention. Such antibody can include a detectable label.
In still another aspect, the invention features several methods, including a screening method for identifying a substance that modulates binding of an XB3 protein to XA21. This method includes the steps of: providing a sample containing the XB3 protein; adding to the sample a candidate substance; adding to the sample XA21; and detecting an increase or decrease in binding of the XB3 protein to XA21 in the presence of the candidate substance, compared to the binding of the XB3 protein to XA21 in the absence of the candidate substance, as an indication that the candidate substance modulates binding of XB3 protein to XA21.
Also within the invention is a method of producing an XB3 protein. This method includes the steps of: providing a cell transformed with an isolated nucleic acid comprising a nucleotide sequence that encodes an XB3 protein; culturing the cell under conditions that allow expression of the XB3 protein; and collecting the XB3 protein from the cultured cell.
Another method within the invention is a screening method for identifying a substance that modulates expression of a gene encoding XB3. This method includes the steps of: providing a test cell; contacting the test cell with a candidate substance; and detecting an increase or decrease in the expression level of the gene encoding XB3 in the presence of the candidate substance, compared to the expression level of the gene encoding XB3 in the absence of the candidate substance, as an indication that the candidate substance modulates the level of expression of the gene encoding XB3.
Yet another method within the invention is a method for isolating a substance that binds XB3. This method includes the steps of: providing a sample of an immobilized XB3; contacting a mixture containing the XB3-binding substance with the immobilized XB3; separating unbound components of the mixture from bound components of the mixture; and recovering the XB3-binding substance from the immobilized XB3 protein. In this method, the XB3-binding substance can be XA21.
Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Commonly understood definitions of molecular biology terms can be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.
By the term xe2x80x9cgenexe2x80x9d is meant a nucleic acid molecule that codes for a particular protein, or in certain cases a functional or structural RNA molecule. For example, the Xb3 gene encodes the XB3 protein.
As used herein, a xe2x80x9cnucleic acidxe2x80x9d or a xe2x80x9cnucleic acid moleculexe2x80x9d means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid). A xe2x80x9cpurifiedxe2x80x9d nucleic acid molecule is one that has been substantially separated or isolated away from other nucleic acid sequences in a cell or organism in which the nucleic acid naturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants). The term includes, e.g., a recombinant nucleic acid molecule incorporated into a vector, a plasmid, a virus, or a genome of a prokaryote or eukaryote. Examples of purified nucleic acids include cDNAs, fragments of genomic nucleic acids, nucleic acids produced polymerase chain reaction (PCR), nucleic acids formed by restriction enzyme treatment of genomic nucleic acids, recombinant nucleic acids, and chemically synthesized nucleic acid molecules. A xe2x80x9crecombinantxe2x80x9d nucleic acid molecule is one made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
By the terms xe2x80x9cXb3 gene,xe2x80x9d xe2x80x9cXb3 polynucleotide,xe2x80x9d xe2x80x9cXb3 nucleic acidxe2x80x9d, or simply xe2x80x9cXb3xe2x80x9d is meant a native XB3-encoding nucleic acid sequence, e.g., the native rice Xb3 cDNA (SEQ ID NO: 1; FIG. 1A), genomic sequences from which Xb3 cDNA can be transcribed, and/or allelic variants and homologs of the foregoing. The terms encompass double-stranded DNA, single-stranded DNA, and RNA.
As used herein, xe2x80x9cproteinxe2x80x9d or xe2x80x9cpolypeptidexe2x80x9d are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation. An xe2x80x9cpurifiedxe2x80x9d polypeptide is one that has been substantially separated or isolated away from other polypeptides in a cell or organism in which the polypeptide naturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants).
By the terms xe2x80x9cXB3 proteinxe2x80x9d xe2x80x9cXB3 polypeptide,xe2x80x9d or simply xe2x80x9cXB3xe2x80x9d is meant an expression product of an Xb3 gene such as the protein of SEQ ID NO:2 (FIG. 1B); or a protein that shares at least 65% (but preferably 75, 80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence identity with SEQ ID NO:2 and displays a functional activity of XB3. A xe2x80x9cfunctional activityxe2x80x9d of a protein is any activity associated with the physiological function of the protein. For example, functional activities of XB3 include ubiquitin ligase activity, the ability to be phosphorylated by XA21, and the ability to specifically bind XA21 in at least one of the in vitro assays described herein.
When referring to a nucleic acid molecule or polypeptide, the term xe2x80x9cnativexe2x80x9d refers to a naturally-occurring (e.g., a xe2x80x9cwild-typexe2x80x9d) nucleic acid or polypeptide. A xe2x80x9chomologxe2x80x9d of a rice Xb3 gene is a gene sequence encoding a XB3 polypeptide isolated from a plant other than rice. Similarly, a xe2x80x9chomologxe2x80x9d of a native XB3 polypeptide is an expression product of an Xb3 homolog.
A xe2x80x9cfragmentxe2x80x9d of an Xb3 nucleic acid is a portion of an Xb3 nucleic acid that is less than full-length and comprises at least a minimum length capable of hybridizing specifically with a native Xb3 nucleic acid under stringent hybridization conditions. The length of such a fragment is preferably at least 15 nucleotides, more preferably at least 20 nucleotides, and most preferably at least 30 nucleotides of a native Xb3 nucleic acid sequence. A xe2x80x9cfragmentxe2x80x9d of an XB3 polypeptide is a portion of an XB3 polypeptide that is less than full-length (e.g., a polypeptide consisting of 5, 10, 15, 20, 30, 40, 50, 75, 100 or more amino acids of native XB3), and preferably retains at least one functional activity of native XB3.
When referring to hybridization of one nucleic to another, xe2x80x9clow stringency conditionsxe2x80x9d means in 10% formamide, 5xc3x97Denhart""s solution, 6xc3x97SSPE, 0.2% SDS at 42xc2x0 C., followed by washing in 1xc3x97SSPE, 0.2% SDS, at 50xc2x0 C.; xe2x80x9cmoderate stringency conditionsxe2x80x9d means in 50% formamide, 5xc3x97Denhart""s solution, 5xc3x97SSPE, 0.2% SDS at 42xc2x0 C., followed by washing in 0.2xc3x97SSPE, 0.2% SDS, at 65xc2x0 C.; and xe2x80x9chigh stringency conditionsxe2x80x9d means in 50% formamide, 5xc3x97Denhart""s solution, 5xc3x97SSPE, 0.2% SDS at 42xc2x0 C., followed by washing in 0.1xc3x97SSPE, and 0.1% SDS at 65xc2x0 C. The phrase xe2x80x9cstringent hybridization conditionsxe2x80x9d means low, moderate, or high stringency conditions.
As used herein, xe2x80x9csequence identityxe2x80x9d means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions. When a subunit position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a given position is occupied by an adenine in each of two DNA molecules, then the molecules are identical at that position. For example, if 7 positions in a sequence 10 nucleotides in length are identical to the corresponding positions in a second 10-nucleotide sequence, then the two sequences have 70% sequence identity. Preferably, the length of the compared sequences is at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).
When referring to mutations in a nucleic acid molecule, xe2x80x9csilentxe2x80x9d changes are those that substitute of one or more base pairs in the nucleotide sequence, but do not change the amino acid sequence of the polypeptide encoded by the sequence. xe2x80x9cConservativexe2x80x9d changes are those in which at least one codon in the protein-coding region of the nucleic acid has been changed such that at least one amino acid of the polypeptide encoded by the nucleic acid sequence is substituted with a another amino acid having similar characteristics. Examples of conservative amino acid substitutions are ser for ala, thr, or cys; lys for arg; gln for asn, his, or lys; his for asn; glu for asp or lys; asn for his or gin; asp for glu; pro for gly; leu for ile, phe, met, or val; val for ile or leu; ile for leu, met, or val; arg for lys; met for phe; tyr for phe or trp; thr for ser; trp for tyr; and phe for tyr.
As used herein, the term xe2x80x9cvectorxe2x80x9d refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as xe2x80x9cexpression vectors.xe2x80x9d
A first nucleic-acid sequence is xe2x80x9coperablyxe2x80x9d linked with a second nucleic-acid sequence when the first nucleic-acid sequence is placed in a functional relationship with the second nucleic-acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein coding regions, in reading frame.
A cell, tissue, or organism into which has been introduced a foreign nucleic acid, such as a recombinant vector, is considered xe2x80x9ctransformed,xe2x80x9d xe2x80x9ctransfected,xe2x80x9d or xe2x80x9ctransgenic.xe2x80x9d A xe2x80x9ctransgenicxe2x80x9d or xe2x80x9ctransformedxe2x80x9d cell or organism (e.g., a plant) also includes progeny of the cell or organism, including progeny produced from a breeding program employing such a xe2x80x9ctransgenicxe2x80x9d cell or organism as a parent in a cross. For example, a plant transgenic for Xb3 is one in which Xb3 nucleic acid has been introduced.
By the term xe2x80x9cXB3-specific antibodyxe2x80x9d is meant an antibody that binds XB3 (e.g., a protein having the amino acid sequence of SEQ ID NO:2), and displays no substantial binding to other naturally occurring proteins other than those sharing the same antigenic determinants as XB3. The term includes polyclonal and monoclonal antibodies.
As used herein, xe2x80x9cbind,xe2x80x9d xe2x80x9cbinds,xe2x80x9d or xe2x80x9cinteracts withxe2x80x9d means that one molecule recognizes and adheres to a particular second molecule in a sample, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample. Generally, a first molecule that xe2x80x9cspecifically bindsxe2x80x9d a second molecule has a binding affinity greater than about 105 to 106 liters/mole for that second molecule.
The term xe2x80x9clabeled,xe2x80x9d with regard to a probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions will control. In addition, the particular embodiments discussed below are illustrative only and not intended to be limiting.